Pleural liquid and kinetic friction coefficient of mesothelium after mechanical ventilation.

Volume and protein concentration of pleural liquid in anesthetized rabbits after 1 or 3h of mechanical ventilation, with alveolar pressure equal to atmospheric at end expiration, were compared to those occurring after spontaneous breathing. Moreover, coefficient of kinetic friction between samples of visceral and parietal pleura, obtained after spontaneous or mechanical ventilation, sliding in vitro at physiological velocity under physiological load, was determined. Volume of pleural liquid after mechanical ventilation was similar to that previously found during spontaneous ventilation. This finding is contrary to expectation of Moriondo et al. (2005), based on measurement of lymphatic and interstitial pressure. Protein concentration of pleural liquid after mechanical ventilation was also similar to that occurring after spontaneous ventilation. Coefficient of kinetic friction after mechanical ventilation was 0.023±0.001, similar to that obtained after spontaneous breathing.

Lubricating recovery of damaged pleural mesothelium: effect of time and of phosphatidylcholines.

Effect of time and phosphatidylcholines (PCs) on lubrication of damaged mesothelium has been investigated. Marked increase in coefficient of kinetic friction (µ) of pleural specimens after mesothelial blotting and rewetting decreased by 23.4±3.5%, 41.8±3.8%, and 40.5±2.7% after 30min, 1h, and 2h. Hence, damaged mesothelium is able to partially reset lubricating molecules on its surface. Increase in µ of post-blotting Ringer 2h after addition of unsaturated PCs (3mg/ml) decreased a little more than after 2h Ringer. Effects of unsaturated and saturated PCs were similar, contrary to expectation raised by their different percentage in pleural and alveolar lavage. Effect of PCs did not increase at 6mg/ml, and was nil at 0.4mg/ml. Increase of µ after short phospholipase treatment decreased by 45.9±2.0% after 2h Ringer, and a little more after addition of unsaturated or saturated PCs. Hence, PCs, as other phospholipids, have a small effect, likely because of difficulty in resetting their relationships with main lubricating molecules.

Pleural mesothelium lubrication after phospholipase treatment.

Coefficient of kinetic friction (µ) of rabbit pleural mesothelium increased after short treatment of specimens with phospholipase C. This increase was removed by addition of a solution with hyaluronan or sialomucin, as previously shown in post-blotting Ringer or after short pronase treatment. After phospholipase µ decreased with increase in sliding velocity, but at highest velocity it was still greater than control; this difference was removed by addition of hyaluronan or sialomucin, as in post-blotting Ringer or after short pronase treatment. Hyaluronan placed on specimen before phospholipase treatment reduced increase in µ by protecting phospholipids from enzyme, as shown by others for alveolar and synovial phospholipids. Samples of parietal pleura stained with silver nitrate showed that mesothelial cells were not disrupted by short phospholipase treatment. Instead, they were disrupted if this treatment was preceded by a short pronase treatment; but even after this disruption addition of hyaluronan or sialomucin brought µ back to control.

Pleural mesothelium lubrication after hyaluronidase, neuraminidase or pronase treatment.

Coefficient of kinetic friction (µ) of pleural mesothelium has been found to increase markedly after mesothelial blotting and rewetting. This increase disappeared after addition of a solution with hyaluronan or sialomucin, though previous morphological studies showed that only sialomucin occurs in mesothelial glycocalyx. In this research we investigated whether µ of rabbit pleural mesothelium increased after hyaluronidase, neuraminidase or pronase treatment. Hyaluronidase and neuraminidase did not increase µ, though neuraminidase cleaved sialic acid from mesothelial glycocalyx of diaphragm specimens, and removed hystochemical stain of sialic acid from glycocalyx. Sialomucin treated with neuraminidase lowered µ of blotted mesothelium, though less than untreated sialomucin; this feature plus lubrication provided by other molecules could explain why µ did not increase after neuraminidase. Short pronase treatment (in order to affect only glycocalyx proteins) increased µ; this increase was removed by hyaluronan or sialomucin. After pronase treatment µ decreased with increase in sliding velocity, indicating a regime of mixed lubrication, as in blotted mesothelium.

Mixed lubrication after rewetting of blotted pleural mesothelium.

Coefficient of kinetic friction (µ) of pleural mesothelium blotted with filter paper, and rewetted with Ringer solution markedly increases; this increase is removed if a sufficient amount of sialomucin or hyaluronan is added to Ringer (Bodega et al., 2012. Respiratory Physiology and Neurobiology 180, 34-39). In this research we found that µ of pleural mesothelium blotted, rewetted, and sliding at physiological velocities and loads, decreased with increase of velocity, mainly at low velocities. Despite this decrease, µ at highest velocity was still double that before blotting. With small concentration of sialomucin or hyaluronan µ was markedly smaller at each velocity, decreased less with increase of velocity, and at highest velocity approached preblotting value. These findings indicate a regime of mixed lubrication in post-blotting Ringer, at variance with boundary lubrication occurring before blotting or postblotting with sufficient macromolecule addition. Greater roughness of mesothelial surface, caused by blotting, likely induces zones of elastohydrodynamic lubrication, which increase with velocity, while contact area decreases.

Lubricating effect of sialomucin and hyaluronan on pleural mesothelium.

Coefficient of kinetic friction (µ) between rabbit visceral and parietal pleura, sliding in vitro at physiological velocities and load, increases markedly after blotting mesothelial surface with filter paper; this increase is only partially reduced by wetting blotted mesothelium with Ringer solution. Given that mesothelial surface is covered by a thick coat with sialomucin and hyaluronan, we tested whether addition of sialomucin or hyaluronan solution after blotting lowers µ more than Ringer alone. Actually, these macromolecules lowered µ more than Ringer, so that µ was no longer significantly higher than its preblotting value. Moreover, Ringer addition, after washout of macromolecule solution, increased µ, in line with their dilution. These findings indicate that mesothelial blotting removes part of these molecules from the coat covering mesothelial surface, and their relevance for pleural lubrication. Transmission electron micrographs of pleural specimens after mesothelial blotting showed that microvilli were partially or largely removed from mesothelium, consistent with a substantial loss of macromolecules normally entrapped among them.

Evidence for Na+-glucose cotransporter in type I alveolar epithelium.

Functional evidence of Na(+)-glucose cotransport in rat lung has been provided by Basset et al. (J. Physiol. 384:325-345, 1987). By autoradiography [(3)H]phloridzin binding has been found confined to alveolar epithelial type II cells in mouse and rabbit lungs (Boyd, J. Physiol. 422: 44P, 1990). In this research we checked by immunofluorescence whether Na(+)-glucose cotransporter (SGLT1) is also expressed in alveolar type I cells. Lungs of anesthetized rats and lambs were fixed by paraformaldehyde, perfused in pulmonary artery, or instilled into a bronchus, respectively. Tissue blocks embedded in paraffin or frozen were sectioned. Two specific anti-SGLT1 antibodies for rat recognizing aminoacid sequence 402-420, and 546-596 were used in both species. Bound primary antibody was detected by secondary antibody conjugated to fluorescein isothiocianate or Texas red, respectively. In some sections cellular nuclei were also stained. In rats alveolar type I cells were identified by fluorescent Erythrina cristagalli lectin. Sections were examined by confocal laser-scanning microscope. Both in rats and lambs alveolar epithelium was stained by either antibody; no labeling occurred in negative controls. Hence, SGLT1 appears to be also expressed in alveolar type I cells. This is functionally relevant because type I cells provide 95-97% of alveolar surface, and SGLT1, besides contributing to removal of lung liquid under some circumstances, keeps low glucose concentration in lining liquid, which is useful to prevent lung infection.

ß2-Adrenergic receptors and G-protein-coupled receptor kinase 2 in rabbit pleural mesothelium.

Former studies on net rate of liquid absorption from small Ringer or 1% albumin-Ringer hydrothoraces in rabbits indicated that Na+ transport and solute-coupled liquid absorption by mesothelium is increased by pleural liquid dilution, and stimulation of ß2-adrenoreceptors (ß2AR). In this research we tried to provide molecular evidence for ß2AR in visceral and parietal mesothelium of rabbit pleura. Moreover, because prolonged stimulation of ß2AR may lead to desensitization mediated by G-protein-coupled receptor kinase 2 (GRK2), we also checked whether GRK2 is expressed in pleural mesothelium. To this end we performed immunoblot assays on total protein extracts from scraped visceral and parietal mesothelium, and from cultured pleural mesothelial cells of rabbits. All three samples showed ß2AR and GRK2 specific bands.

Na+-glucose cotransporter is also expressed in mesothelium of species with thick visceral pleura.

Molecular evidence for Na+-glucose cotransporter (SGLT1) in rabbit pleural mesothelium has been recently provided, confirming earlier functional findings on solute-coupled liquid absorption from rabbit pleural space. In this research we checked whether SGLT1 is also expressed in pleural mesothelium of species with thick visceral pleura, which receives blood from systemic circulation, but drains it into pulmonary veins. To this end immunoblot assays were performed on total protein extract of scraped visceral and parietal mesothelium of lambs and adult sheep, and of a human mesothelial cell line. All of them showed SGLT1 specific bands. Moreover, confocal immunofluorescence images of lamb pleural mesothelium showed that SGLT1 is located in apical membrane. Therefore, a solute-coupled liquid absorption should also occur from pleural space of species with thick visceral pleura. Because of this protein-free liquid entering interstitium between visceral mesothelium and capillaries, inherent Starling forces should be different than hitherto considered, and visceral pleura capillaries could absorb liquid even in these species.

Pleural liquid and its exchanges.

After an account on morphological features of visceral and parietal pleura, mechanical coupling between lung and chest wall is outlined. Volume of pleural liquid is considered along with its thickness in various regions, and its composition. Pleural liquid pressure (P(liq)) and pressure exerted by lung recoil in various species and postures are then compared, and the vertical gradient of P(liq) considered. Implications of lower P(liq) in the lung zone than in the costo-phrenic sinus at iso-height are pointed out. Mesothelial permeability to H(2)O, Cl(-), Na(+), mannitol, sucrose, inulin, albumin, and various size dextrans is provided, along with paracellular "pore" radius of mesothelium. Pleural liquid is produced by filtration from parietal pleura capillaries according to Starling forces. It is removed by absorption in visceral pleura capillaries according to Starling forces (at least in some species), lymphatic drainage through stomata of parietal mesothelium (essential to remove cells, particles, and large macromolecules), solute-coupled liquid absorption, and transcytosis through mesothelium.

Expression of Na+-glucose cotransporter (SGLT1) in visceral and parietal mesothelium of rabbit pleura.

Indirect evidence for a solute-coupled liquid absorption from rabbit pleural space indicated that it should be caused by a Na(+)/H(+)-Cl(-)/HCO(3)(-) double exchanger and a Na(+)-glucose cotransporter [Agostoni, E., Zocchi, L., 1998. Mechanical coupling and liquid exchanges in the pleural space. In: Antony, V.B. (Ed.), Clinics in Chest Medicine: Diseases of the Pleura, vol. 19. Saunders, Philadelphia, pp. 241-260]. In this research we tried to obtain molecular evidence for Na(+)-glucose cotransporter (SGLT1) in visceral and parietal mesothelium of rabbit pleura. To this end we performed immunoblot assays on total protein extracts of scraped visceral or parietal mesothelium of rabbits. These showed two bands: one at 72kDa (m.w. of SGLT1), and one at 55kDa (which should also provide Na(+)-glucose cotransport). Both bands disappeared in assays in which SGLT1 antibody was preadsorbed with specific antigen. Molecular evidence for Na(+)/K(+) ATPase (alpha1 subunit) was also provided. Immunoblot assays for SGLT1 on cultured mesothelial cells of rabbit pleura showed a band at 72kDa, and in some cases also at 55kDa, irrespectively of treatment with a differentiating agent. Solute-coupled liquid absorption hinders liquid filtration through parietal mesothelium caused by Starling forces, and favours liquid absorption through visceral mesothelium caused by these forces.

Pleural liquid during hemorrhagic hypotension.

The effect of approximately 25% or 35% blood loss (b.l.) on volume, pressure, and protein concentration of pleural liquid has been determined in anesthetized rabbits in lateral or supine posture. Volume and pressure of pleural liquid did not change with 25% b.l. 30 and 60 min after beginning of hemorrhage, and with 35% b.l. at 30 min (bleeding time approximately 10 and 12 min, respectively). With 35% b.l. protein concentration of pleural liquid was 85% greater (P<0.01) than control; moreover, percent albumin was smaller (P<0.05), and percent globulin greater (P<0.05) than control. Decrease in arterial plasma protein concentration, hematocrit, and pH after hemorrhage fit literature data. Ventilation at 15 and 30 min increased (P<0.01) by 16% and 23%, respectively, with 25% b.l., but it did not change with 35% b.l., a condition borderline to survival in anesthetized rabbits without ad hoc treatment. Pleural liquid seems protected against derangements from hemorrhage up to 25% b.l. for periods shorter than 1 h.

Distribution and mixing of a liquid bolus in pleural space.

Distribution and mixing time of boluses with labeled albumin in pleural space of anesthetized, supine rabbits were determined by sampling pleural liquid at different times in various intercostal spaces (ics), and in cranial and caudal mediastinum. During sampling, lung and chest wall were kept apposed by lung inflation. This was not necessary in costo-phrenic sinus. Here, 10 min after injection, lung inflation increased concentration of labeled albumin by 50%. Lung inflation probably displaces some pleural liquid cranio-caudally, increasing labeled albumin concentration caudally to injection point (6th ics), and decreasing it cranially. Boluses of 0.1-1 ml did not preferentially reach mediastinal regions, as maintained by others. Time for an approximate mixing was approximately 1 h for 0.1 ml, and approximately 30 min for 1 ml. This relatively long mixing time does not substantially affect determination of contribution of lymphatic drainage through stomata to overall removal of labeled albumin from 0.3 ml hydrothoraces lasting 3 h [Bodega, F., Agostoni, E., 2004. Contribution of lymphatic drainage through stomata to albumin removal from pleural space. Respir. Physiol. Neurobiol. 142, 251-263].

Contribution of lymphatic drainage through stomata to albumin removal from pleural space.

The contribution of lymphatic drainage through the stomata of parietal mesothelium to the overall removal of labeled albumin from the pleural space was found 89% in sheep with very large hydrothoraces (10 ml/kg), a condition involving a approximately 20 times increase in lymphatic drainage [Broaddus et al., J. Appl. Physiol. 64 (1988) 384]. We determined this contribution in anesthetized rabbits with small (0.12 ml/kg) and large (2.4 ml/kg) hydrothoraces of Ringer-albumin with labeled albumin and labeled dextran-2000 kDa. This dextran was used as marker of liquid removal through the stomata because it should essentially leave the pleural space through the stomata only, owing to its size. The removal of labeled albumin by lymphatic drainage through the stomata was 39% of the overall removal in the small hydrothoraces, and 64% in the large ones. Hence, lymphatic drainage through the stomata does not contribute most of protein and liquid removal from the pleural space under physiological conditions, as it has been maintained. It markedly increases with the increase in pleural liquid volume.

Labeled albumin in plasma and removal paths from pleural space in control and increased ventilation.

Increased ventilation was shown to markedly increase lymphatic drainage and plasma content of labeled proteins injected into pleural space relative to control ventilation. These proteins reach plasma by lymphatic drainage: directly through parietal pleura stomata, and indirectly from pleural interstitium, reached by diffusion, convection and transcytosis. Increased drainage from interstitium should not involve a comparable increase in protein removal from pleural space by these transports, while increased drainage through stomata involves a comparable increase in protein removal. Hence, relative increase in labeled protein removal from pleural space caused by increased ventilation should be marked only if drainage through stomata contributed most of this removal, whereas relative increase of labeled proteins in plasma should be marked in either case. We injected 3 ml of albumin-Ringer with albumin-Texas red into the pleural space of three groups of anesthetized rabbits: control, CO2-, or muscle stimulation-increased ventilation. Increased ventilation for 3 h (78 and 61%, respectively) increased (P < 0.01) labeled albumin in plasma by 132 and 106%, respectively, but did not significantly increase its removal. Hence, lymphatic drainage through stomata should not contribute most of liquid and protein removal from pleural space.

Albumin transcytosis from the pleural space.

Occurrence of transcytosis in pleural mesothelium was verified by measuring removal of labeled macromolecules from pleural liquid in experiments without and with nocodazole. To this end, we injected 0.3 ml of Ringer-albumin with 750 microg of albumin-Texas red or with 600 microg of dextran 70-Texas red in the right pleural space of anesthetized rabbits, and after 3 h we measured pleural liquid volume, labeled macromolecule concentration, and, hence, labeled macromolecule quantity in the liquid of this space. Labeled albumin left was 318 +/- 28 microg in control and 419 +/- 17 microg in nocodazole experiments (means +/- SE); hence, whereas ventilation was similar its removal was greater (P < 0.01) in control experiments. Labeled dextran left was 283 +/- 10 microg in control and 381 +/- 21 microg in nocodazole experiments; hence, whereas ventilation was similar its removal was greater (P < 0.01) in control experiments. These findings indicate occurrence of transcytosis from the pleural space. Liquid removed by transcytosis was 0.05 ml/h. This amount times unlabeled albumin concentration under physiological conditions (10 mg/ml) times lumen-vesicle partition coefficient for albumin (0.78) provides fluid-phase albumin transcytosis: approximately 203 microg. h(-1) kg(-2/3). Transcytosis might contribute a relevant part of protein and liquid removal from the pleural space.

Albumin transcytosis in mesothelium.

Apparent permeability to albumin (P(alb)) was measured with (125)I-albumin in specimens of rabbit parietal pericardium from lumen to interstitium (L-I) and from interstitium to lumen (I-L). With albumin concentration (C(alb)) 0.5%, P(alb) (x 10(-5) cm/s) L-I at 37 degrees C was 0.172 +/- 0.019 SE; it decreased to 0.092 +/- 0.022 I-L at 37 degrees C, 0.089 +/- 0.021 L-I at 12 degrees C, and 0.084 +/- 0.018 I-L at 12 degrees C. These findings provide evidence for an active transport L-I, likely transcytosis. With C(alb) 2.5%, 0.05%, and 0.005%, P(alb) L-I at 37 degrees C was 0.188 +/- 0.023, 0.156 +/- 0.021, and 0.090 +/- 0.021, respectively; at 12 degrees C it was 0.089 +/- 0.017, 0.083 +/- 0.019, and 0.087 +/- 0.026, respectively. Hence, active albumin transport ceases with C(alb) 0.005%; P(alb) values I-L at 12 degrees C and with C(alb) 0.005% are similar and provide diffusional permeability. With physiological C(alb) (approximately 1%), active albumin flux was approximately 5 x 10(-4) micromol x h(-1) x cm(-2). Apparent permeability to FITC-dextran 70 (P(dx)) was also measured. P(dx) (x 10(-5) cm/s) L-I at 37 degrees C with C(alb) 0.5% was 0.095 +/- 0.018; it decreased to 0.026 +/- 0.004 I-L (37 degrees C, C(alb) 0.5%), 0.038 +/- 0.007 at 12 degrees C (L-I, C(alb) 0.5%), 0.030 +/- 0.009 with C(alb) 0.005% (L-I, 37 degrees C), and 0.032 +/- 0.011 with nocodazole (L-I, 37 degrees C, C(alb) 0.5%). These findings provide evidence for transcytosis and confirm conclusions drawn from P(alb). Vesicular liquid flow, computed from vesicular dextran flux (fluid-phase only), was approximately 3.5 microl x h(-1) x cm(-2). Transcytosis seems a relevant mechanism, removing protein and liquid from serous cavities.